Plant-based production of biopharmaceuticals
Rainer Fischer1,2, Eva Stoger1, Stefan Schillberg2, Paul Christou2 and
Richard M Twyman3,4
Plants are now gaining widespread acceptance as a general
platform for the large-scale production of recombinant proteins.
The first plant-derived recombinant pharmaceutical proteins are
reaching the final stages of clinical evaluation, and many more
are in the development pipeline. Over the past two years, there
have been some notable technological advances in this
flourishing area of applied biotechnology, as shown by the
continuing commercial development of novel plant-based
expression platforms. There has also been significant success in
tackling some of the limitations of plant bioreactors, such as low
yields and inconsistent product quality, that have limited the
approval of plant-derived pharmaceuticals.
Addresses
1
Institute for Molecular Biotechnology, Biology VII, RWTH Aachen,
Worringerweg 1, 52074 Aachen, Germany
2
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME),
Grafschaft, Auf dem Aberg 1, 57392 Schmallenberg, Germany
3
Department of Biology, University of York, Heslington,
York YO10 5DD, UK
4
e-mail: [email protected]
Current Opinion in Plant Biology 2004, 7:152–158
This review comes from a themed issue on
Plant biotechnology
Edited by Pal Maliga and Ian Graham
1369-5266/$ – see front matter
ß 2004 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2004.01.007
Abbreviations
CaMV cauliflower mosaic virus
ER
endoplasmic reticulum
GUS
b-glucuronidase
TSP
total soluble protein
Introduction
Proteins can be used as diagnostic reagents, vaccines and
drugs, and this creates a strong demand for the production of recombinant proteins on an industrial scale.
Commercial protein production has traditionally relied
on microbial fermentation and mammalian cell lines, but
these systems have disadvantages in terms of cost, scalability and safety that have prompted research into
alternatives. Despite industry inertia and conservatism,
plants have emerged as one of the most promising general production platforms for tomorrow’s biologics. Plants
allow the cost-effective production of recombinant proteins on an agricultural scale, while eliminating risks of
product contamination with endotoxins or human pathoCurrent Opinion in Plant Biology 2004, 7:152–158
gens [1,2,3,4]. Another advantage of the use of plants in
recombinant protein production is that vaccine candidates can be expressed in edible plant organs, allowing
them to be administered as unprocessed or partially
processed material [5].
Current limitations of plant bioreactor technology include
the low yields that are achieved for many proteins (which
are often caused by poor protein stability), difficulties
with downstream processing (leading to inconsistent product quality), and the presence of non-authentic glycan
structures on recombinant human proteins. These problems raise regulatory issues and have prevented the
routine approval of plant-derived biopharmaceuticals
for use in clinical trials [6,7]. In this review, we discuss
technical advances achieved over the past two years that
have helped to address these limitations, thus bringing
the prospect of affordable, plant-derived biologics
another step closer.
Emerging production platforms for
biopharmaceuticals
Choosing a host species
Many of the early, plant-derived recombinant proteins
were produced in transgenic tobacco plants and were
extracted directly from harvested leaves. The continuing popularity of tobacco reflects its status as a wellestablished expression host for which robust transformation procedures and well-characterized regulatory
elements for the control of transgene expression are
available [1]. Furthermore, its high biomass yields and
rapid scalability make tobacco very suitable for commercial molecular farming. It is also a non-food, nonfeed crop, and so carries a reduced risk of transgenic
material or recombinant proteins contaminating feed
and human food chains [8]. Tobacco has been adopted
as a platform system by several biotech companies,
including Planet Biotechnology Inc. (http://www.
planetbiotechnology.com/) and Meristem Therapeutics
(http://www.meristem-therapeutics.com/), the only two
companies to have plant-derived pharmaceuticals undergoing phase-II clinical trials.
One disadvantage of tobacco is its high content of nicotine and other toxic alkaloids, which must be removed
completely during downstream processing steps. Although
low-alkaloid tobacco cultivars are available, attention
has turned to other leafy crops for pharmaceutical production. These crops include lettuce, which has been
used for clinical trials with a hepatitis B virus subunit
vaccine [9], and alfalfa, which is being promoted as a
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Plant-based production of biopharmaceuticals Fischer et al. 153
platform system by Medicago Inc. (http://www.medicago.
com). This Canadian biotech company has isolated novel
promoters that allow high-level protein expression in
alfalfa leaves, and has focussed on the early part of the
production pipeline by developing alfalfa cell-culture
and transient-expression technology [10]. Advantages of
alfalfa include its high biomass yield and the fact that it is
a perennial plant that fixes its own nitrogen. A strong
advantage of alfalfa for pharmaceutical production is that
fact that glycoproteins synthesised in alfalfa leaves tend
to have homogeneous glycan structures, which is important for batch-to-batch consistency (see review by
Gomord in this issue). However, alfalfa is a feed crop
and its leaves contain large amounts of oxalic acid, which
might interfere with processing.
Although leafy crops are advantageous in terms of biomass yield, proteins that are expressed in leaves tend to
be unstable, which means the harvested material has a
limited shelf life and must be processed immediately
after harvest. By contrast, proteins that are expressed in
cereal seeds are protected from proteolytic degradation;
they can remain stable for up to three years at room
temperature (E Stoger, unpublished data) and for at least
three years at refrigerator temperature without significant
loss of activity [11]. Several different cereals, including
rice, wheat, barley and maize, have been investigated as
potential hosts for recombinant protein production [8,12].
Maize has been chosen by Prodigene Inc. (http://
www.prodigene.com), an industry leader in cereal-based
commercial protein production, because it has a high
biomass yield, because it is easily transformed and
manipulated in vitro, and because the production of
transgenic maize can be scaled up conveniently. Maize
has been used for the commercial production of the
technical proteins avidin and b-glucuronidase (GUS)
[13,14]. In addition, Prodigene is exploring its use for
the production of subunit vaccines [15], recombinant
antibodies [16] and further technical enzymes, such as
aprotinin and laccase [17].
Although it is beneficial to focus on a small number of
platform technologies for the bulk production of biopharmaceuticals, the delivery of recombinant vaccines in
edible plant organs is exceptional because it would be
advantageous to use locally grown plants for vaccination
campaigns. Therefore, a variety of different expression
hosts have been evaluated. Potato was the first major
system to be used for vaccine production, and transgenic
potato tubers have been administered to humans in at
least three clinical trials to date [18]. Over the past year,
potatoes have been evaluated for the production of human
serum albumin [19], novel vaccine candidates [20,21],
tumour necrosis factor a (TNF-a) [22] and antibodies
[23,24]. Other production hosts that have been used to
express vaccines include tomatoes, bananas, carrots, lettuce, maize, alfalfa, white clover and Arabidopsis.
www.sciencedirect.com
Oilcrops are useful hosts for protein production because
the oil bodies can be exploited to simplify protein
isolation. An example is the oleosin-fusion platform
developed by SemBioSys Genetics Inc. (http://www.
sembiosys.com/), in which the target recombinant protein is expressed in oilseed rape or safflower as a fusion
with oleosin (see below; [P1]). The Finnish biotech
company UniCrop (http://www.unicrop.fi/) is also developing an oilseed technology platform, although in this
case, the idea is to isolate recombinant proteins from the
rapidly developing sprouts cultivated in bioreactors.
Finally, there have been significant recent developments
in the use of more diverse plant species, which can easily
be contained, propagated and transformed, to produce
recombinant proteins. Mayfield et al. [25] have
described a protein expression system that is based on
the unicellular green alga Chlamydomonas reinhardtii. In
this system, chloroplast-targeted transgenes were used to
express an antibody that recognised herpes simplex virus
glycoprotein D (see article by Franklin and Mayfield in
this issue). Other simple plants that have been adopted as
bioreactors include Lemna (duckweed), which is being
developed as a platform technology by Biolex Inc. (http://
www.biolex.com; [26]), and the moss Physcomitrella patens,
which is being developed by Greenovation Inc., Freiburg,
Germany (http://www.greenovation.com; see article by
Decker and Reski in this issue). The advantages and
disadvantages of different expression hosts are summarised in Table 1.
Alternative plant-based expression systems
The majority of plant-derived recombinant pharmaceutical proteins have been produced by nuclear transformation and the regeneration of transgenic plant lines,
followed by the extraction and purification of proteins
from the transgenic tissues. Although nuclear gene transfer is now routine in many species, it has disadvantages
in terms of production time-scales, which are being
addressed or circumvented by the development of alternative plant-based production technologies (Table 2).
Transient expression is generally used to verify transformation construct activity and to validate small amounts of
recombinant protein. It can be achieved by the vacuum
infiltration of leaves with recombinant Agrobacterium tumefaciens, resulting in the transient transformation of many
cells [27]. High levels of protein expression are achieved
for a short time, but generally the technique is insufficient
for commercial-scale production [28,29]. Recently, however, several reports have described how this agroinfiltration process could be scaled-up more efficiently.
Baulcombe and colleagues [30] have shown that the
loss of protein expression seen after a few days is predominantly caused by gene silencing. They managed to
increase the expression levels of several proteins at least
50-fold by co-expressing the p19 protein from tomato
Current Opinion in Plant Biology 2004, 7:152–158
154 Plant biotechnology
Table 1
Plant expression hosts used for biopharmaceutical production.
Species
Model plants
Arabidopsis thaliana
Simple plants
Physcomitrella patens,
Chlamydomonas reinhardtii, Lemna
Leafy crops
Tobacco
Alfalfa, clover
Lettuce
Cereals
Maize, rice
Advantages
Disadvantages
Range of available mutants,
accessible genetics, ease of transformation
Not useful for commercial
production (low biomass)
Containment, clonal propagation, secretion into
medium, regulatory compliance, homologous
recombination in Physcomitrella
Scalability
High yield, established transformation and expression
technology, rapid scale-up, non-food/feed
High yield, useful for animal vaccines, clonal
propagation, homogenous N-glycans (alfalfa)
Edible, useful for human vaccines
Low protein stability in harvested
material, presence of alkaloids
Low protein stability in harvested
material, presence of oxalic acid
Low protein stability in harvested material
Wheat, barley
Protein stability during storage, high yield, easy
to transform and manipulate
Protein stability during storage
Legumes
Soybean
Economical, high biomass, expression in seed coat
High protein content
Low yields, difficult to transform
and manipulate
Low expression levels, difficult to
transform and manipulate
Low expression levels
Pea, pigeon pea
Fruits and vegetables
Potato, carrot
Tomato
Edible, proteins stable in storage tissues
Edible, containment in greenhouses
Potato needs to be cooked
More expensive to grow, must be
chilled after harvest
Oilcrops
Oilseed rape, Camelina sativa
Oleosin-fusion platform, sprouting system
Lower yields?
bushy stunt virus, a known inhibitor of gene silencing.
Furthermore, researchers at Medicago Inc. have described how the agroinfiltration of alfalfa leaves can be
scaled up to 7500 leaves per week, producing micrograms
of recombinant protein each week [10]. Similarly, we
have shown that up to 100 kg of wildtype tobacco leaves
could be processed by agroinfiltration, resulting in the
production of several hundred milligrams of protein (R
Fischer, S Schillberg, unpublished).
Another emerging tobacco transient-expression technology is based on the use of plant viruses as expression
vectors. Virus-infected plants have been used to produce
several pharmaceutical proteins, including vaccine candidates and antibodies, one of which is now undergoing
phase-I clinical trials [31]. The advantages of virusbased production include the rapid onset of expression,
the systemic spread of the virus so that recombinant
protein is produced in every cell, and the fact that
more than one vector can be used in the same plant,
allowing multimeric proteins to be assembled [32]. Plant
virus expression systems are discussed by Gleba et al.
in this issue.
The tobacco chloroplast transgenic system is another
promising variant, which was boosted this year by the
Table 2
Comparison of different plant-based production systems.
System
Advantages
Disadvantages
Transgenic plants, accumulation within plant
Yield, economy scalability, establishment
of permanent lines
Containment, purification
Yield, multiple gene expression,
low toxicity, containment
Yield, timescale, mixed infections
Timescale
Timescale, containment, secretion into
medium (purification), regulatory compliance
Production timescale, regulatory compliance
Transgenic plants, secretion from roots or leaves
Transplastomic plants
Virus-infected plants
Agroinfiltration
Cell or tissue culture
Current Opinion in Plant Biology 2004, 7:152–158
Scale, yield, cost of production facilities
Absence of glycosylation, some evidence
of horizontal gene transfer
Biosafety, construct-size limitations
Cost
Cost
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Plant-based production of biopharmaceuticals Fischer et al. 155
launch of a new company, Chlorogen, to capitalise on its
pharmaceutical potential (http://www.chlorogen.com).
Transplastomic plants are generated by introducing
DNA into the chloroplast genome, usually by particle
bombardment [33,34]. The advantages of chloroplast
transformation are many: the transgene copy number is
high because of the many chloroplasts in a typical photosynthetic cell, there is no gene silencing, multiple genes
can be expressed in operons, the recombinant proteins
accumulate within the chloroplast thus limiting toxicity to
the host plant, and the absence of functional chloroplast
DNA in the pollen of most crops provides natural transgene containment.
The chloroplast transgenic system has achieved remarkably high expression levels, recently exceeding 25%
total soluble protein (TSP) for a tetanus toxin fragment
[35], 11% TSP for human serum albumen [36] and 6%
TSP for a thermostable xylanase [37]. At present,
chloroplast transformation is routine only in tobacco
and C. reinhardtii (see above and the article by Franklin
and Mayfield, this issue). However, plastid transformation has been achieved in a growing number of plant
species, including carrot and tomato [33,34]. The ability
to transform the chromoplasts of fruit and vegetable
crops has obvious advantages for the expression of
subunit vaccines [38].
Plant cell cultures can be used for the production of smallmolecule drugs, but they are also advantageous for molecular farming because of the high level of containment
that they offer and the possibility of producing proteins
under current good manufacturing practice (cGMP) conditions [39]. Tobacco suspension cells are the most popular system at present, although pharmaceutical proteins
have also been produced in soybean, tomato and rice
cells, and in tobacco hairy roots [40–43]. More than 20
pharmaceutical proteins have been produced in plant
cell-suspension cultures, including antibodies, interleukins, erythropoietin, human granulocyte-macrophage colony stimulating factor (hGM-CSF) and hepatitis B
antigen [39]. Unfortunately, few of these proteins have
been expressed at yields sufficient for commercial production. As discussed below, the problem of poor yields
could be addressed in part by the use of optimised regulatory elements. For example, the expression of hGMCSF in rice suspensions using an inducible promoter
produced far greater yields than was possible using
tobacco cells and a constitutive promoter [41].
Strategies to improve protein yields
The factors that affect recombinant protein yields in
transgenic plants and other plant systems have recently
been reviewed in detail [44]. The general approach is to
maximise both the efficiency of all stages of gene expression and protein stability by appropriate subcellular
targeting.
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Transgene expression in plants used for molecular farming is often driven by the strongest available constitutive
promoters. However, regulated promoters are increasingly used, particularly those that allow external regulation by physical or chemical stimuli [45]. Several novel
inducible promoters that may be useful in molecular
farming applications have been described recently. For
example, a peroxidase gene promoter isolated from sweet
potato (Ipomoea batatas) was used to drive the gusA reporter gene in transgenic tobacco. This promoter produced
30 times more GUS activity than did the cauliflower
mosaic virus (CaMV) 35S promoter following exposure
to hydrogen peroxide, wounding or ultraviolet light [46].
The wounding response is interesting as it would allow
post-harvest induction of gene expression in the same
manner as the CropTech mechanical gene activation
(MeGA) system, which is based on a tomato hydroxy3-methylglutaryl CoA reductase2 (HMGR2) promoter.
A novel seed-specific promoter from the common bean
(Phaseolus vulgaris) has been used to express a singlechain antibody in Arabidopsis thaliana. In contrast to the
CaMV 35S promoter, which resulted in antibody accumulation to 1% TSP, the bean arc5-I promoter resulted in
antibody levels in excess of 36% TSP in homozygous
seeds, and the antibody retained its antigen binding
activity and affinity [47]. A trichome-specific promoter
that might be useful for the secretion of recombinant
proteins into the leaf guttation fluid has also been
described in tobacco [48]. Another secretion system,
which is being commercialised by Phytomedics Inc.
(http://www.phytomedics.com), involves the secretion
of recombinant proteins into tobacco root exudates and
the leaf guttation fluid. This was developed for the
production of human secreted alkaline phosphatase and
has recently been used for the secretion of recombinant
antibodies [49].
Subcellular targeting plays an important role in determining the yield of recombinant proteins because the compartment in which a recombinant protein accumulates
strongly influences the interrelated processes of folding,
assembly and post-translational modification. Comparative targeting experiments with full-size immunoglobulins and single-chain fragment variable (scFv) fragments
have shown that the secretory pathway is more suitable
for folding and assembly than the cytosol, and is therefore
an advantageous site for high-level protein accumulation
[50]. Antibodies that are targeted to the secretory pathway
using either plant or animal amino-terminal signal peptides usually accumulate to levels that are several orders
of magnitude greater than those of antibodies expressed
in the cytosol. Occasional exceptions to this general
observation suggest that the intrinsic features of each
antibody might also contribute to overall stability [51].
The endoplasmic reticulum (ER) provides an oxidising
environment and an abundance of molecular chaperones
Current Opinion in Plant Biology 2004, 7:152–158
156 Plant biotechnology
but few proteases. These features are likely to be the most
important factors affecting protein folding and assembly.
It has been shown recently that antibodies that are targeted to the secretory pathway in transgenic plants interact specifically with the molecular chaperone BiP [52].
In the absence of further targeting information, proteins
that accumulate in the secretory system are secreted to
the apoplast. Depending on its size, the protein can be
retained in the apoplast or might leach from the cell, with
important implications for production systems that are
based on cell-suspension cultures. The stability of antibodies in the apoplast is lower than that in the lumen of
the ER. Therefore, antibody expression levels can be
increased even further if the protein is retrieved to the
ER lumen using an H/KDEL carboxy-terminal tetrapeptide tag [53]. Accumulation levels of proteins tagged in
this way are generally 2–10-fold greater than those of
identical proteins that lack the KDEL signal [44]. ER
retention can also influence the structure of glycan chains
on plant-derived proteins ([54,55] and our unpublished
observations), but we do not discuss the post-translational modification of plant-derived proteins any further
because this topic is treated in detail by Gomord and
Faye in this issue. Targeting is especially important if the
recombinant protein is toxic to the production host. For
example, the accumulation of avidin in the cytosol of
transgenic tobacco plants is toxic, but plants can be
regenerated successfully when this molecule is targeted
to the vacuole [56].
Downstream processing
Although high-level expression is necessary to provide
good yields in plant-based production systems, the efficient recovery of recombinant proteins must also be
optimised. Secretion systems are advantageous because
no disruption of plant cells is necessary during protein
recovery; hence, the release of phenolic compounds is
avoided. Nevertheless, the recombinant proteins may be
unstable in the culture medium. The use of affinity tags to
facilitate the recovery of proteins is a useful strategy as
long as the tag can be removed after purification to restore
the native structure of the protein. In the oleosin fusion
system mentioned earlier, the fusion protein can be
recovered from oil bodies using a simple extraction procedure and the recombinant protein separated from its
fusion partner by endoprotease digestion [P1]. Similarly,
we have devised a strategy in which recombinant proteins
are expressed as fusion constructs that contain an integral
membrane-spanning domain derived from the human Tcell receptor, and are then purified from membrane fractions [57]. Recent strategies that have been described
include the expression of His-tagged GUS-fusion proteins in tobacco chloroplasts [58], the extraction of Histagged proteins by foam fractionation [59], and the release
of recombinant proteins using a modified intein expression system [60].
Current Opinion in Plant Biology 2004, 7:152–158
Conclusions
Plants have many advantages over established production
technologies for the large-scale expression of recombinant proteins, but several challenges remain to be addressed in terms of improving yields and product quality.
A small number of plant-derived biologics are approaching commercialisation, but these are the minority that
have met the technological challenges, cleared the regulatory hurdles and overcome inertia in the biotechnology
industry. We are facing a growing demand for protein
therapeutics and diagnostics, but the capacity to meet
those demands using established facilities is lacking. A
shift to plant bioreactors might therefore become necessary within the next few years, making it more imperative
that these issues are addressed and solved.
Acknowledgements
The authors would like to thank their colleagues at the Fraunhofer IME,
RWTH Aachen and other institutes for their contribution to the field of
molecular farming. Funding from the European Community project
PharmaPlant is gratefully acknowledged.
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